Team:Montpellier/Perspectives

Engineered microbes are poised to be the next generation of therapeutics working in the human body. Synthetic biology researchers aim at developing tools to reach this goal in future years. Our project fits into such research. However, we are still in the early days of what a bacteria-driven contraception could be. Hence the need for more research about this subject that we will be discussed in this section.

Next experiments

For our project we imagined and designed a lot of experiments that we wanted to do. We were not able to conduct all of them because of lack of time. Here we present what would be the next steps of our project.

Measuring spermicides production and efficiency

Antibodies

The produced antibodies could be purified to check if they are produced and secreted. We have confirmed that the antibodies are produced in E. coli. Purification with the E-tag on the antibodies could give information about the quantity that bacteria produce and secrete. The experiment with the purified antibodies on sperm could tell which quantity is needed to fully inhibit sperm motility which is very important for the future of the project.

Another important thing about the antibodies is that the exact mechanisms of their actions are not known yet. Understanding how the antibodies work and where they are fixed on the sperm could be interesting, and should be compared to the literature [1].

Peptides

The experiments detailed on the sperm motility assay could be applied to other peptides. We designed sequences for lacticin and subtilosin but other peptides could be studied such as nisin for example. The sequences could be designed and cloned, as we did for LL-37, and the products tested on sperm as we did for nisin. Those experiments could tell which ones are the easiest to produce and the most efficient.

Another interesting experiment would be to test the efficiency of each protein according to their concentration, like it has been done for nisin [2]. The results of the experiment could be compared with experiments showing which molecules is easiest to produce and would help determine the best candidate. Other experimental procedures that are important for choosing the right protein are to study the production and stability over time, the interaction between the spemicidal product and the flora, and also the potential interactions with the human immune system. Ultimately, the constructs should be transformed in L. jensenii, and tested for expression and secretion as described above. Then, bacteria secreting the different spermicides would be incubated with sperm and their effect on motility would be measured. If these experiments were successful, one could envision inoculating these genetically engineered L. jensenii on mice and test their effect on the animals’ reproduction.

Next steps of the project

For our project, a lot of additional steps are necessary before being able to create a contraception method. We present here some of the next steps that should be done to continue the project.

First, one important matter to keep in mind is that in our case we worked on mice sperm (the reasons are discussed here). But for contraception, all products should be tested on human sperm. The peptides and antibodies that we designed and used have been described to be working for human spermatozoa.

Genetic stability, safety, environmental dispersal, and reversibility

The genes expressing the spermicidal molecules should in the future be integrated into the bacterial chromosome. This could limit horizontal gene transfers of the spermicidal coding sequence Furthermore, because antibiotic resistances are banned for in vivo applications (guidelines FDA) in absence of antibiotic selection, there is a high probability that the bacteria gets rid of the plasmid and inserting the sequences in the chromosome could avoid this.

Moreover, a major aspect of this project and the main concern of people is how to stop the contraceptive effect and make sure there will not be any dissemination. We worked on those aspects throughout bibliography research. They are a lot of mechanisms already existing and that are possible to use for this application [3][4][5]. In the future, the use of multiple mechanisms, including auxotrophic, and kill-switches would probably be the best answer to this problem. The next step would be to search which mechanisms are usable in Lactobacilli and test them in a vaginal like environment.

Going beyond contraception: Lactobacillus jensenii as a platform for vaginal
microbiota engineering

Finally, we think it is important to mention that the work on Lactobacillus jensenii should not be restricted to contraception application. Indeed, the work on the vaginal flora could lead to different kind of projects. Research about therapeutic applications of the vaginal microbiome engineering already exist. In particular, L. jensenii has been shown to be a promising chassis for preventing sexually transmitted diseases (STDs), for example by secreting anti-HIV molecules [6][7]. With that in mind, we started developing an open-source toolbox for L. jensenii that we hope can help other laboratories to work with the strain for different applications.

We believe that our work is a first stone to the study of the vaginal microbiota and the development of a new contraceptive method. We hope that this work will be pursued in Montpellier and by other laboratories to permit the further development of our project.

Popularizing contraception, the microbiota and synthetic biology

During our project, we realized that the topic of contraception was still carrying a lot of taboo. We also found that the general public has little knowledge about the microbiota, especially the vaginal one, and on synthetic biology. We started several projects to address these issues, including a comic book, and we are planning to continue this work by organizing events in collaborations with various artists. Two events in November are scheduled so we can continue on our way to popularization of our project (see the Education and Public Engagement page for more details). We are hoping that our project and initiatives could help people knowing more about their body and what scientists are doing for it. We also hope that our project can inspire other teams and laboratories to study the vaginal microbiota for contraception or other applications.

References
[1]	Samuel, A. S., & Naz, R. K. (2008). Isolation of human single chain variable fragment antibodies against specific sperm antigens for immunocontraceptive development. Human reproduction, 23(6), 1324-1337.
[2]	Aranha, C., Gupta, S., & Reddy, K. V. R. (2004). Contraceptive efficacy of antimicrobial peptide Nisin: in vitro and in vivo studies. Contraception, 69(4), 333-338.
[3]	Bojar, D., Scheller, L., Charpin-El Hamri, G., Xie, M., & Fussenegger, M. (2018). Caffeine-inducible gene switches controlling experimental diabetes. Nature Communications, 9(1), 2318.
[4]	Rovner, A. J., Haimovich, A. D., Katz, S. R., Li, Z., Grome, M. W., Gassaway, B. M., ... & Isaacs, F. J. (2015). Recoded organisms engineered to depend on synthetic amino acids. Nature, 518(7537), 89.
[5]	Mandell, D. J., Lajoie, M. J., Mee, M. T., Takeuchi, R., Kuznetsov, G., Norville, J. E., ... & Church, G. M. (2015). Biocontainment of genetically modified organisms by synthetic protein design. Nature, 518(7537), 55.
[6]	Marcobal, A., Liu, X., Zhang, W., Dimitrov, A. S., Jia, L., Lee, P. P., ... & Lagenaur, L. A. (2016). Expression of human immunodeficiency virus type 1 neutralizing antibody fragments using human vaginal Lactobacillus. AIDS research and human retroviruses, 32(10-11), 964-971.
[7]	Liu, X., Lagenaur, L. A., Simpson, D. A., Essenmacher, K. P., Frazier-Parker, C. L., Liu, Y., ... & Lee, P. P. (2006). Engineered vaginal lactobacillus strain for mucosal delivery of the human immunodeficiency virus inhibitor cyanovirin-N. Antimicrobial agents and chemotherapy, 50(10), 3250-3259.